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9 October 2005. MARCKS, the myristoylated alanine-rich C kinase substrate, has been studied
in non-neuronal cells for years as a regulator of cell shape and motility.
A few researchers have noted alterations in MARCKS gene expression or
protein phosphorylation in suicide victims or people with bipolar disorder (McNamara et al., 1999; Pandey et al., 2003) and in Alzheimer patients (Kimura et al., 2000). The protein has also been fingered as a primary
target, via protein kinase C (PKC), of lithium and other drugs used to
treat bipolar disorder.

One place MARCKS has been spotted is in dendritic spines, but what it is doing there has been a mystery. That has changed with a new report from Barbara Calabrese and Shelly Halpain from the Scripps Research Institute in La Jolla, California. In a paper published this week in Neuron, Calabrese and Halpain show that MARCKS functions in the maintenance and remodeling of dendritic spines in cultured hippocampal neurons. Their work also reveals that protein kinase C-catalyzed phosphorylation of MARCKS leads to dendritic spine remodeling, giving the protein a starring role in synaptic plasticity, learning and memory.

MARCKS knockout mice die around birth, so Calabrese and Halpain turned to cultured neurons to investigate the effects of MARCKS knockdown or overexpression. When the scientists expressed RNAi for MARCKS in 3-week-old cultures of rat hippocampal neurons, they observed a reduction in the density, width, and length of dendritic spines. But overexpression of MARCKS also caused a reduction in spine number, with increased length. From this, the authors concluded that MARCKS dynamically regulates spine stability and morphology, and this regulation is finely tuned.

MARCKS is a multitalented protein that binds to membranes (via a myristoyl moiety) and F-actin (via the effector domain), serving as a bridge between the cell surface and the actin cytoskeleton. When the effector domain gets phosphorylated by protein kinase C, MARCKS falls off the membrane, and its binding to actin is disrupted. To dissect the role of each domain, the researchers overexpressed mutant MARCKS proteins and carefully observed spine morphology and actin dynamics. Mutations in the effector domain that abolished PKC phosphorylation sites, or changed them to aspartic acid to mimic phosphorylation, both resulted in decreases in spine number, but in somewhat different ways. The unphosphorylatable mutant caused an increase in spine length, similar to that seen with overexpression of wild-type MARCKS. The opposite mutation, pseudophosphorylated MARCKS, induced a morphology similar to the knockdown, with reduced width and length. The different effects of the mutants reflected their different subcellular distributions: wild-type and nonphosphorylatable MARCKS were mostly membrane-associated, while the pseudophosphorylated protein was mainly cytosolic.

A look at the cytoskeletal rearrangements in these cells showed that the pseudophosphorylated MARCKS enhanced actin clustering in the spine head and reduced head motility. Decreased motility is often seen after glutamate receptor activation, raising the possibility that MARCKS phosphorylation by PKC could be responsible for real-life regulation of spine plasticity. Indeed, when the researchers treated neurons with the PKC activator phorbol ester, they recapitulated the loss of spines achieved with the pseudophosphorylated mutant, and this loss was prevented by expression of the nonphosphorylatable MARCKS.

It came as a bit of a surprise that loss of spines elicited by MARCKS mutant proteins was not associated with synapse loss. The same number of synapses seemed to be redistributed onto the remaining spines, and the synapses were functionally equivalent to those in normal cells as indicated by unchanged postsynaptic excitatory currents. These results show that even in the midst of MARCKS-influenced spine changes, synaptic function is maintained.

Dendritic spines put the plastic in synaptic plasticity—they proliferate during long-term potentiation and retrench during long-term depression. The identification of MARCKS as a player in this process jibes with another recent report implicating MARCKS in learning and memory in mouse hippocampus (McNamara et al., 2005), and should lead the way to more understanding of its involvement in AD and other neuropathologies.—Pat McCaffrey (Alzheimer Research Forum).

The formation of dendritic spines during development and their structural plasticity in the adult brain are critical aspects of synaptogenesis and synaptic plasticity. Actin is the major cytoskeletal source of dendritic spines, and polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Some, but not all, postmortem studies in schizophrenia have identified reduced dendritic spine density in neurons of the hippocampal formation and dorsolateral prefrontal cortex (for review, see Honer et al., 2000); however, little is known about the underlying pathogenic mechanisms affecting synaptic function in the disease.

Many different factors and proteins are known to control dendritic spine development and remodeling (see Ethell and Pasquale, 2005). Comprehensive investigation of the effectors and signaling pathways involved in regulating actin dynamics may provide insight into the molecular mechanisms mediating altered cortical microcircuitry in the disease.

David Lewis and colleagues have previously reported reduced spine density in the basilar dendrites of pyramidal neurons in laminar III of the DLPFC (though this is not clearly a laminar-specific finding). In their current study, Hill et al. extended these investigations to examine gene expression levels for members of the RhoGTPase family of intracellular signaling molecules (e.g., Cdc42, Rac1, RhoA, Duo), and Debrin, an F-actin binding protein, all of which are critical signal transduction molecules involved in spine formation and maintenance. Their aim was to determine whether alterations in the expression of one of more molecules may underlie the reduced spine density seen in the disorder. Hill et al. report that reductions in Cdc42 and Duo mRNA are observed in the DLPFC in schizophrenia and correlate with spine density on deep layer III pyramidal neurons. This paper provides preliminary evidence that "gene expression levels of certain mRNAs encoding proteins known to be key regulators of dendritic spines are reduced in the DLPFC in schizophrenia." However, the paper also reports that these two mRNAs are reduced in lamina where significant reductions in spine density are not observed in schizophrenia. These results may suggest, as the authors discuss, that reduced expression of Cdc42 and Duo might contribute to, but is not sufficient to cause reduced, spine density.

Synaptic dysfunction has received increasing attention as a key feature of schizophrenia’s neuropathology and possibly its genetic etiology (Law et al., 2004). Neuregulin 1 (NRG1), a lead schizophrenia susceptibility gene, is known to be a critical upstream regulator of signal transduction pathways modulating cytoskeletal dynamics, playing pivotal roles in synapse formation and function. We have previously reported that isoform-specific alterations of the NRG1 gene and its primary receptor, ErbB4, are apparent in the brain in schizophrenia and related to genetic risk for the disease (Law et al, 2005a, Law et al, 2005b). Altered NRG1/ErbB4 signaling in schizophrenia may be a pathway to aberrant cortical neurodevelopment and synaptic function via dysregulation of specific intracellular signaling pathways linked to actin. The lack of significant alterations in gene expression levels for proteins such as Rac1 and RhoA in the DLPFC (gray matter, as reported by Hill and colleagues) in schizophrenia might be because the primary defect may not lie with the expression of these molecules but with the upstream modulation of their function and activity. Therefore, investigation of the proteins themselves, their phosphorylation status and activity, will be useful in understanding how genes effect molecular pathways that mediate biological risk for schizophrenia. The study of intracellular signaling cascades may be a route to a closer understanding of the biological mechanisms underpinning the association of genes such as NRG1 and ErbB4 with schizophrenia and their relationship to its neuropathology.